Theses and Dissertations from UMD
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Item STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES(2016) Rowland, Sara; Robb, Frank T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.Item ROLE OF SALT BRIDGES IN GROEL ALLOSTERY(2014) Yang, Dong; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chaperonin GroEL facilitates protein folding with two stacked back-to-back, identical rings and the "lid", co-chaperonin GroES. The mis-folded/unfolded substrate protein (SP) adjusts the chaperonin cycling from an asymmetric to a symmetric cycle by catalyzing the release of ADP from the trans ring of GroEL, thus promoting the R to T allosteric transition. ATP binding to the SP bound ring promotes the association of a second GroES and subsequently a GroEL-GroES 2 "football" complex is formed as the folding functional form. However, ADP does release spontaneously, albeit at very slow rate, in the absence of SPs. The intrinsic mechanism by which GroEL relaxes to the lower potential energy T state remains poorly understood. A network of salt bridges forms and breaks during the allosteric transitions of GroEL. Residue D83 in the equatorial domain forms an intra-subunit salt bridge with K327 in the apical domain, and R197 in the apical domain forms an inter-subunit salt bridge with E386 in the intermediate domain. These two salt bridges stabilize the T state and break during the T to R state transition. Removal of these salt bridges by mutation destabilizes the T state and favors the R state of GroEL. These mutations do not alter the intrinsic ATPase activity of GroEL. However, the affinity for nucleotides becomes enhanced and ADP release is hindered such that SP cannot displace the equilibrium to the T state, as normally it does in the wild type. The exchange of ADP to ATP and association of a second GroES is compromised with the following GroEL-GroES 2 "football" formation is hindered. These mutations do not completely eliminate the T state, in the absence of nucleotide, as shown biochemically and by crystal structures. The biased allosteric equilibrium hampers the formation of folding active "football" complex as the mutant GroEL's incompetency to revisit T state in the presence of nucleotide, but not due to the elimination of its T state. This study revealed the critical role of salt bridges in regulating the allosteric transitions of GroEL and conjugated formation of the "football" complex.Item An Analysis of the Stability, Aggregation Propensity, and Negative Cooperativity of the Escherichia coli Chaperonin GroEL(2013) Wehri, Sarah; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Since the discovery of chaperonin GroEL and co-chaperonin GroES, there has been a deluge of literature investigating many aspects of the system. Substrate proteins are protected from aggregation through a cycle of capture, encapsulation, and release made possible through rigid body motions of the GroE system driven by a combination of allosteric controls influenced by nucleotide, potassium and denatured protein termed substrate protein (SP). This dissertation first explores the sequential transition of GroEL that maintains the rings operating in an alternating fashion. To do this, an intra-subunit, inter-domain mutant, GroELD83AT state. Steady state ATPase assays, stopped-flow fluorescence, and gel filtration chromatography were all used to demonstrate that the trans ring must access the T state before ligands can be discharged from the cis ring. The dual-heptameric ring structure of GroEL and the post-translational assembly of the protein make creating mutants with a mutation within a single subunit of a ring almost impossible, however the ability to do so opens the opportunity for a myriad of experiments that explore the allosteric transitions of GroEL. Two potential recombination methods, acetone treatment and heat treatment, were investigated. Förster resonance energy transfer (FRET) and electrospray ionization mass spectrometry (ESI-MS) were used to study recombination facilitated by such treatments. Recombination using the acetone method resulted in a one-in-one-out subunit exchange, however aggregation complicated the exchange. Heat treatment resulted in exchange of rings. Finally, dynamic light scattering (DLS) was used to investigate stability and aggregation on the chaperonin. It was observed that the chaperonin is stable for over 30 days while incubated continuously at 37°C in sterile buffered solution, however interesting aggregation kinetics are observed upon addition of acetone, the solvent used to strip SP from GroEL during the standard lab purification procedure. GroEL partitions into 10nm and 100nm species that are extremely stable before the appearance of macromolecular aggregates and precipitation is observed.